The patent's assignee is
News editors obtained the following quote from the background information supplied by the inventors: "The following is offered as background information only and is not admitted to be prior art to the present invention.
"Cancer cells are characterized by an ability to proliferate indefinitely and to invade normal tissue cells surrounding a cancerous tumor. In addition, many types of cancer cells can metastasize throughout the body whereby the tumor may be disseminated in the cancer patient's body.
"The mechanism of cancer metastasis is presumed as follows: (1) cancer cells proliferate in a primary cancer colony; (2) blood vessels are newly formed; (3) the malignant cancer cells infiltrate and penetrate the newly formed blood vessels; (4) the cancer cells circulate within the human body; (5) the cancer cells reach a target organ; (6) the cancer cells extravasate from blood vessels; (7) the cancer cells proliferate in the target organ; and (8) a metastatic focus is formed.
"The cure rate of malignant cancer tumors has increased with early diagnosis and with the progress of therapies, but metastases of malignant tumors are often beyond current therapies. Chemotherapies are used to arrest metastases following removal of tumors but often with unsatisfactory results. It is therefore desirable to develop more effective inhibitors against malignant tumor metastases.
"Conventional methods for treating cancer have also increased the survival and quality of life for cancer patients. Such conventional methods include surgery, for example, removal of the prostate gland (radical prostectomy), radiotherapy, and chemotherapy. In addition, bone marrow transplantation is becoming useful in treating patients with certain types of cancers.
"Combination therapies may be used in treating cancer and are typically not addictive. In some cases, cross effects and treatment load can result in a lower effectiveness for the combinations, than either treatment alone.
"Other treatments such as radiation, while useful for a wide range of cancers, does not typically result in a complete cure. Given the severity of many cancers and the mortality rate, a drug may be thought of as successful if it improves the quality of life by delaying the growth of tumors, or if it prolongs life without actually curing the condition. In many circumstances, an individual is treated with a specific composition or with a combination of therapies that can eliminate from about 90 to about 95% of the malignant cells, but the remaining cells can re-grow and metastasize, ultimately resulting in death.
"LIM kinase 1 (LIMK 1) is one of the regulatory proteins that modulate the actin cytoskeleton by inactivating an actin-binding protein, cofilin, through addition of a phosphate group to cofilin. Actin cytoskeleton is maintained by the constant severing and joining of small units of actin and any deviation away from this normal dynamic may lead to abnormal behavior of a particular cell. LIMK1 plays an important role in maintaining cell architecture through actin cytoskeleton. As an important regulator of cell behavior, levels of LIMK1 need to be regulated in cells for the maintenance of normal cellular function. The concentration of LIMK1 has previously been found at increased levels in some cancer cells; for example, cancer cells that are highly aggressive and capable of causing metastasis or the spread of tumors in mice. Research has shown that a partial inhibition of LIMK 1 synthesis using antisense RNA in these cells resulted in the inhibition of cell growth and more specifically, regression of the invasive property of these cells.
"Efforts to identify compositions which inhibit the expression of LIMK 1 and, which therefore, should be useful in the treatment and prevention of cancer cell metastasis, has led to the use of the 'antisense' or non-encoding LIM Kinase 1, which exhibits an ability to inhibit the expression of LIMK 1 in certain cancer cells.
"Antisense RNA mediated gene therapy is a widely used method of gene inactivation and is suitable for gene therapy use. However, RNA mediated gene inactivation does not work for all genes, and may not be used as a global method of gene inactivation."
As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventor's summary information for this patent application: "Before explaining the disclosed embodiment of the present invention in detail it is to be understood that the invention is not limited in its application to the details of the particular arrangement shown since the invention is capable of other embodiments. Also, the terminology used herein is for the purpose of description and not of limitation.
"In one embodiment, the present invention provides, for example, a method for treating cancers and individuals at risk of developing cancers by administering an antisense RNA of LIM Kinase 1.
"In another embodiment, the present invention indicates that the expression of LIM kinase, one of the proteins that modulates actin dynamics, is over expressed in denocarcinomatous prostatic epithelium and cancer cell lines.
"In one embodiment of the present invention, there is provided an advantageous therapy for inhibiting the metastasis of cancers cells, including, for example, breast, ovarian, lung, and prostate cancer cells. More specifically, the invention, in embodiments relates to the use of LIM kinase 1 antisense RNA to reduce the synthesis of endogenous LIM kinase 1 in prostate cancer cell lines. This reduction has been shown to reduce the number of metastizing cancer cells.
"In another embodiment of the invention, a partial reduction of LIMK1 altered cell proliferation by arresting cells at the G2/M phase of the cell cycle. Data has also shown a changed cell shape and the associated inhibition of the invasive tendencies of metastatic prostate cancer cells. Ectopic expression of LIMK1 promotes the acquisition of the invasive phenotype by benign prostate epithelial cells. Experimental data provides evidence of a novel role of LIMK1 in regulating cell division and in regulating the invasive properties of prostate cancer cells.
"In yet another embodiment of the invention, results indicate that the invasive properties of prostate cancer cells is not mediated by phosphorylation of cofilin. Experimental data correlates with the recent observations showing a metastasis-associated chromosomal gain of 7q11.2 in prostate cancer, suggesting a possible gain in LIMK1 DNA (7q11.23).
"Yet another embodiment of the instant invention comprises partial amino acid and nucleotide sequences of LIMK1.
"In still another embodiment of the present invention results indicate that the antisense RNA of LIM kinase 1 can be used successfully in the functional inactivation of LIM kinase 1.
"In other embodiments of the present invention, antisense RNA may be used, for example, to inhibit gene function and to demonstrate that the LIMK1 gene may find use, for example, as a therapeutic target for developing effective cancer drugs.
"In yet another embodiment results indicated, for the first time, that LIMK 1 may find use as a novel target for anti sense RNA mediated gene inactivation. Results also indicate that the expression of LIMK 1 may be used as a predictive marker for prostate cancer cell metastasis.
BRIEF DESCRIPTION OF THE DRAWINGS
"In a further effort to make the above-recited features and aspects of the specific embodiments of the invention, clear, more specific descriptions of the invention briefly summarized above may be had by reference to embodiments thereof which are illustrated in the drawings. These drawings form a part of the specification. It is to be noted, however, that the drawings illustrate embodiments of the invention and therefore are not to be considered limiting in their scope.
"FIG. 1a shows an immunoblot analysis of LIMK1 in total lysates of human prostate cell lines.
"FIGS. 1b, 1c and 1d show the expression of LIMK1 of normal/benign areas in prostrate tissues.
"FIGS. 1e, 1f, and 1g show the expression of LIMK1 of cancerous glands in prostrate tissues.
"FIGS. 1b and 1e show hematoxylin/eosin-stained slides at 100.times.. magnification.
"FIGS. 1d and 1g show normal and cancerous tissues at 200.times. magnification. Arrows in FIGS. 1c and 1d indicate light staining in basal cells in normal/benign areas. Arrows in FIGS. 1f and 1g indicate intense staining in cells in cancerous areas. Cell lines used were PrEC and prostate epithelial cells in glyceraldehyde-3-phosphate dehydrogenase, GAPDH.
"FIGS. 2a and 2b show the LIMK1 concentration in BPH-1 and PC3 cells before, FIG. 2a and after FIG. 2b immunoprecipitation with anti-LIMK1 antibody. IB immunoblot. Sup, supernatant.
"FIG. 2c represents the in vitro phosphorylation of recombinant cofilin by LIMK1 immunoprecipitated from PC3 cells.
"FIG. 2d shows in vivo phosphorylation of cofilin in BPH-1 and PC3 cells, and is an Immunoblot analysis of cell lysates before immunoprecipitation and shows the initial concentration of cofilin in both samples in the bottom panel of FIG. 2d.
"FIG. 2e shows the immunoblot analysis of phosphorylated cofilin or cofilin in crude cell extracts using anti-phospho-cofilin or anti-cofilin antibodies.
"FIGS. 3a and 3b show the RNA blot analysis of LIMK1 in PC3ASL (a) and PC3LacZ (b) cells induced successively (+) or not (-).
"FIG. 3c shows the concentration of LIMK1 in transfected PC3 cells harvested at different time points after successive induction.
"FIG. 3d shows the concentration of phosphorylated cofilin (P-cofilin) or cofilin in transfected PC3 cells after continuous induction. Despite the reduced concentration of LIMK1 no noticeable difference in phospho-cofilin concentration was detected in PC3ASL cells Using a Western blot in glyceraldehyde-3-phosphate dehydrogenase GAPDH. LIMK1 was effectively reduced in PC3ASL cells by expression of antisense LIMK1, but there was no concurrent reduction in cofilin phosphorylation.
"FIG. 4a depicts a [.sup.3H]thymidine incorporation in PC3ASL and PC3LacZ cells induced successively (1) or not (N). Data represents values relative to the initial time point (0 hours) and as the mean.+-.S.E of three independent experiments.
"FIGS. 4b, 4c, 4d shows the flow cytometric analysis of cell cycle transition in synchronized PC3LacZ (FIG. 4c) and PC3ASL (FIG. 4d) cells successively induced for 72 hours. Cells were arrested at the G.sub.1/S phase boundary and released. FIG. 4b indicates the percentage of cells at different stages of the cell cycle.
"FIGS. 4c and 4d show cell cycle profiles of similar progression of PC3ASL and PC3LacZ cells to the S and G.sub.2/M phase up to 6 hours after release, but only the PC3LacZ cells of FIG. 4c continue to the G.sub.1 and S phase, whereas the PC3AL cells of FIG. 4d undergo a G.sub.2/M arrest from about 10 hours onwards. The reduced expression of LIMK1 in PC3 cells is associated with the suppression of cell growth and cell cycle arrest in the G.sub.2/M phase.
"FIG. 5a shows transfected cells that were successively induced for from about 46 to about 50 hours before being plated into the invasion chambers. Ectopic expression of LIMK1 in BPH-1 cells increased invasion significantly after induction with ponasterone A, for from about 23 to about 25 hours, with about a 100% difference in invasion in different cell lines. Data represents the mean.+-.S.E. of quantitative analysis of three independent assays (each from two different clones of transfected PC3 and BPH-1 cells).
"FIGS. 5b and 5c, are bright field images of cells invaded to the underside of the control and Matrigel membranes.
"FIG. 5 d presents staining of LIMK1 in BPHL and BPHLacZ cells showing predominantly cytoplasmic distribution of LIMK1 in BPHL cells. Nuclei were stained with 4,6-diamidino-2-phenylindole at 200.times. magnification. The Reduced expression of LIMK1 is associated with a reversion of the invasion ability of PC3 cells.
"FIGS. 6a and 6b shows phase-contrast images of PC3LacZ, (FIG. 6a) and PC3ASL cells, (FIG. 6b) after successive induction for 72 h.
"FIGS. 6c, 6d, 6e, 6f, 6g and 6h, show dual staining of continuously induced PC3LacZ cells (FIGS. 6c, 6e, and 6g) and PC3ASL cells, (FIGS. 6d, 6f, and 6h) with Alexa 488-conjugated phalloidin and anti-LIMK1, (FIGS. 6c and 6d), anti-paxillin, (FIGS. 6e and 6f,) or MT1-MMP, (FIGS. 6g and 6h) antibodies. Arrows indicate localization of LIMK1 in lamellipodia along with actin in FIG. 6c. Arrows in FIG. 6d indicate accumulation of LIMK1 between actin stress fibers and cell boundary. The arrows in FIG. 6e indicate localization of paxillin to the small adhesion points at the lamellipodia along with actin. The redistribution of paxillin and accumulation in large areas between actin stress fibers and cell periphery is show in FIG. 6f, and localization of MT1-MMP (also in the enlarged section) and actin to the lamellipodia is shown in FIG. 6g. Scale bar, 10 .mu.m.
"FIGS. 6i and 6j, indicate phase contrast images of BPHLacZ, 6i and BPHL, 6j showing the difference in growth pattern. Here, reduced expression of LIMK1 is associated with changes in cell morphology, cytoskeleton organization, and the invasive ability of PC3 cells."
For additional information on this patent application, see: Chakrabarti, Ratna. Methods and Compositions for Inhibiting the Proliferation of Cancer Cells. Filed
Keywords for this news article include: Antibodies, Antisense Technology, Biotechnology, Drugs, Anions, Genetics, Oncology, Treatment, Immunology, Phosphates, Lim Kinases, Chemotherapy, Cytoskeleton, Radiotherapy, Therapeutics, Bioengineering, Blood Proteins, Immunoglobulins, Phosphoric Acids, Cancer Gene Therapy, Cellular Structures, Intracellular Space.
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